44 STUDIES ON ALKALINE PHOSPHATASE 



TABLE IV 



The effect of washing sections of various thicknesses for various lengths 

 of time. The sections were of rat kidney incubated with glycerophos- 

 phate by the Gomori method, after which calcium phosphate present in 

 the section was converted into cobalt phosphate by treatment with co- 

 balt nitrate. The sections were then washed in 0.002 percent sodium 

 veronal. + indicates that the section is washed free of cobalt nitrate 

 but has lost no appreciable amount of cobalt phosphate. 



stituted for the cobalt solution, and the sections are washed 

 in dilute acetic acid or citric acid instead of in distilled water, 

 the yolk could be freed from stain without dissolving the pre- 

 cipitated phosphate. 



The Physiological Sites of Phosphatase Activity- 

 It has been demonstrated that the various cytochemical re- 

 actions do indicate sites of alkaline phosphatase in the section. 

 It still remains to be shown that the enzyme in a fixed prepara- 

 tion does not diffuse to a significant degree during or after 

 fixation. Neumann has demonstrated that practically all the 

 phosphatase may be removed from some materials by prolonged 

 contact with 30 percent alcohol. Clearly, exposure to dilute 

 alcohol must be avoided if diffusion of phosphatase is not to 

 cause serious difficulty. So far as I am aware, Martin and 

 Jacoby (1949) were the first to obtain clear evidence that 

 phosphatase may diffuse during the course of incubation. There 

 are at least three possible methods of estimating the extent to 

 which the distribution of phosphatase is modified by diffusion. 

 In all these methods it is necessary to use a logarithmic series of 

 incubation times. The exact lengths of time of incubation must 

 be chosen according to the amount of phosphatase which is 

 present in the section. 



